7KHO

NicA2 variant N462V in complex with (S)-nicotine

Summary for 7KHO

• Entry DOI: 10.2210/pdb7kho/pdb
• Related: 7KHN
• Descriptor: Amine oxidase, (S)-3-(1-METHYLPYRROLIDIN-2-YL)PYRIDINE, FLAVIN-ADENINE DINUCLEOTIDE, ... (4 entities in total)
• Functional Keywords: flavoenzyme, oxidoreductase, nicotine, rossmann-core fold, flavoprotein
• Biological source: Pseudomonas putida S16
• Total number of polymer chains: 4
• Total formula weight: 218392.82

Authors

Tararina, M.A.,Allen, K.N. (deposition date: 2020-10-21, release date: 2021-02-10, Last modification date: 2023-10-18)

Primary citation

Tararina, M.A.,Dam, K.K.,Dhingra, M.,Janda, K.D.,Palfey, B.A.,Allen, K.N.
Fast Kinetics Reveals Rate-Limiting Oxidation and the Role of the Aromatic Cage in the Mechanism of the Nicotine-Degrading Enzyme NicA2.
Biochemistry, 60:259-273, 2021

PubMed Abstract: In , the flavoprotein nicotine oxidoreductase (NicA2) catalyzes the oxidation of ()-nicotine to -methyl-myosmine, which is nonenzymatically hydrolyzed to pseudooxynicotine. Structural analysis reveals a monoamine oxidase (MAO)-like fold with a conserved FAD-binding domain and variable substrate-binding domain. The flavoenzyme has a unique variation of the classic aromatic cage with flanking residue pair W427/N462. Previous mechanistic studies using O as the oxidizing substrate show that NicA2 has a low apparent of 114 nM for ()-nicotine with a very low apparent turnover number ( of 0.006 s). Herein, the mechanism of NicA2 was analyzed by transient kinetics. Single-site variants of W427 and N462 were used to probe the roles of these residues. Although several variants had moderately higher oxidase activity (7-12-fold), their reductive half-reactions using ()-nicotine were generally significantly slower than that of wild-type NicA2. Notably, the reductive half-reaction of wild-type NicA2 is 5 orders of magnitude faster than the oxidative half-reaction with an apparent pseudo-first-order rate constant for the reaction of oxygen similar to . X-ray crystal structures of the N462V and N462Y/W427Y variants complexed with ()-nicotine (at 2.7 and 2.3 Å resolution, respectively) revealed no significant active-site rearrangements. A second substrate-binding site was identified in N462Y/W427Y, consistent with observed substrate inhibition. Together, these findings elucidate the mechanism of a flavoenzyme that preferentially oxidizes tertiary amines with an efficient reductive half-reaction and a very slow oxidative half-reaction when O is the oxidizing substrate, suggesting that the true oxidizing agent is unknown.

PubMed: 33464876
DOI: 10.1021/acs.biochem.0c00855

Experimental method

X-RAY DIFFRACTION (2.69 Å)